06 September 2006
Myoglobin and Hemoglobin
Dr. John David Dignam, Ph.D.

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Functions of Myoglobin and Hemoglobin

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• Proteins with similar functions, structure, and origins
• Mb = myoglobin
  • Solubility of O2 in water is 35 μM
    • Not very soluble, and requires a carrier
  • Mb increases O2 available in muscle
    • [Mb] in red muscle = 500 μM (8g/L)
    • Capacity of O2 = 500 μM
      • That’s over 10 times O2’s solubility in water
• Hb = hemoglobin
  • Hemoglobin carries O2 from capillaries in lungs to capillaries in lungs to capillaries in tissues
  • [Hb] in red blood cell = 4,000 μM (300g/L)
  • Capacity for O2 = 16,000 μM
    • Increases effective solubility of O2 by a lot!
• Almost all vertebrates and invertebrates have hemoglobin
  • Champsocephalus gunnari, the ice fish, has no hemoglobin

Heme

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• Contains Fe-protophyrin IX ring
  • Carbon ring with 4 nitrogen atoms bound to Fe
  • Oxidized Fe+++ will not bind oxygen
  • Reduced Fe++ binds oxygen
    • Systems in blood maintain iron in Fe++ state

Myoglobin

• Monomer
• Single heme which is the O2 binding site
• Single O2 site dominated by α helix
  • 80-85% α helix
• Heme is held between 2 histidines and some hydrophobic residues
  • Help normally by hydrophobic interactions
  • Not covalently attached so if Mb is denatured, the heme falls off
  • O2 binds partly directly to iron and partly with one of the histidines

Hemoglobin

• Essentially looks like 4 subunits which looks like myoglobin
• Tetramer of (α2β2)
  • Can be dissociated into dimers
• Four oxygen binding sites
• Effector sites: protons, 2,3-BPG, CO¬2
  • 2,3-bis-phosphoglyceride regulates O2 affinity
• Interactions holding heme to hemoglobin is mostly hydrophobic, just like Mb

O2 Saturation for Myoglobin

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• Calculate O2 concentration by adjusting partial pressure
• Y = fractional saturuation
  • Y = 1 means all of it is saturated
• Myoglobin has a hyperbolic binding pattern
  • Bound oxygen binds quickly at first, but eventually saturates
• Protein-Ligand <--> P + L
  • Kd = Dissociation Constant
  • Kd = ( [P] x [L] ) / [PL]
  • [Pt] = [P] + [PL]
  • [PL] = ( [Pt] x [L] ) / ( Kd + [L] )

Binding Isotherms

• Ligand and acceptor form a complex in equilibrium with free acceptor and ligand
• Concentration of the complex depends on concentration of ligand and equilibrium constant (Kd)

Fractional Saturuation

• Y = [PL] / [Pt]
• Y = L / ( Kd + [L] )
• Effect of [O2] on Y
  • Y = [O2] / ( Kd + [O2] )
  • When O2 equals 0, Y = 0
  • When O2 equals Kd, Y = 0.5
    • 1/2 Vmax
  • When O2 >>> Kd, Y approaches 1

Determining Kd

• Determine [PL] at several concentrations of [L]
• Use graphic or computer method to estimate Kd
• Several graphic methods
  • Hyperbolic saturation plots
  • Linear transformations of binding equation
• Apply linear or non-linear least squares fit of function to data to get estimates of Kd

Scatchard Plot

• Y = mx + b
  • ( [PL] / [L]) = ( -1 / Kd ) x [PL] + ( [Pt] / Kd )
    • Y = [PL] / [L]
    • m = ( -1 / Kd)
    • X = [PL]
    • b = ( [Pt] / Kd )

Double Reciprocal Equation

• Y = mx + b
  • ( 1 / [PL] ) = ( Kd / [Pt] ) x (1 / [L] ) + (1 / [Pt] )
    • Y = ( 1 / [PL] )
    • m = ( Kd / [Pt] )
    • X = (1 / [L] )
    • b= (1 / [Pt] )

Multiple Binding Sites

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• PL <--> P+L
  • K1 = [P] x [L] / [PL]
• PL2 <--> PL + L
  • K2 = [PL] x [L] / [PL2]
• If sites are independent and non-interacting, K1 = K2
• If there is positive cooperativity, K1 > K2
• If there is negative cooperativity, K1 < K2
• Lower affinity with K is higher

Adair Equation

• Y = Ka[X] + KaKb[X]2 / 1+ 2Ka[X]+Ka¬Kb[X]2
• Protein with 2 binding sites where Ka and Kb are association constants for two different sites
• Association constant is the inverse of a dissociation constant

Cooperativity and Allosterism

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• Hill approximation for ligand binding to multiple sites
• n is the Hill coefficient corresponding to the minimum number of sites
  • Y = [L]n / Kn + [L]n
  • Or, alternatively, Y = [L]n / K’ + [L]n

Properties of Hill Equation

• Equation assumes only unliganded and fully liganded protein
• n = Hill coefficient, does not need to be an integer
• Determination of n gives measure of cooperativitiy
  • n=1, no cooperativity
    • e.g. myoglobin
  • n>1, positive cooperativity
    • Ligand binding at the first site makes binding at the second site easier
    • Characterized as sigmoidal
  • n<1, negative cooperativity
    • Ligand binding at the first site makes binding at the second site more difficult

Hill Equation in Linear Form

• Y = [O2]n / Kdn + [O2]n
• Log Y / (1-Y) = n Log [O2] – n Log Kd
  • Y = Log Y
  • m = n
  • X = Log [O2]
  • b = -n Log Kd

Myoglobin vs. Hemoglobin

• Mb n = 1
  • Mb shows no cooperativity
  • O2 binds tightly and retained by Mb
  • Parabolic curve
• Hb n = 2.8
  • Hb shows strong cooperativity
  • Hb doesn’t have high binding affinity for O2 because it needs to take up and eventually release O2
  • Sigmoidal curve

Monod Model

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• Concerted model for positive coopertivity
• Hb has two conformational states:
  • R – relaxed, high affinity state
  • T – Taut, low affinity state
    • Has a cavity where a ligand can bind and stabilize the low affinity T state
    • Binds 2, 3 BPG
• Conformers are symmetric (T4 or R4)
  • All subunits either in T or R state
• Symmetry due to subunit contacts
  • Change the conformation of one subunit forces change in conformation of all to retain shape of Hb
• Low affinity T and high affinity R states exist in equilibrium

R and T States

• Both R and T states can bind O2
• Specific subunit contacts stabilizes T state
  • O2 binding disrupts contacts in T state and promotes the formation of the R state
• Low O2 favors the T state
• Distribution of R and T states exist in the absence of O2; O2 just changes the distribution
• O2 binding must be symmetrical – either none or all subunits are bound

Koshland Sequential Model

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• Absence of ligand, protein is in one conformation
  • Equalibrium of 2 states, R and T, but not present without the ligand
• Symmetry is no required (RT states can occur)
• Sequential changes in conformation
• Ligand binding induces T --> R transition
• Ligand subunit can influence the conformation of its neighboring subunit
  • Influence affinity for ligand to increase or decrease in affinity
    • Can account for both positive and negative behavior
    • Monod model cannot account for negative behavior easily

O2-Induced Structural Changes

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• Changes occur after the first 2 sites of Hb are bound
• Fe moves 0.06 nm into the plane of the heme
  • Heme Iron is displaced into the ring of the heme by O2 binding
• His on F8 helix moves with the iron
• C=O of Val FG5 looses H-bonding contact with Tyr HC2
• C-terminus is displaced
• Diminished subunit interactions
  • Alters ionic interactions through salt bridges
  • Networks of ionic interactions is altered
• Structural changes result in the cooperative nature of O2 binding

Conserved Residues in Hb

• His Fe
  • Heme contact (proximal His)
• His E7
  • O2 binding site (distal His)
• Phen CD1
  • Heme contact
• Leu F4
  • Heme contact
• Gly B6
  • Allows B and E helices to contact
• Pro C2
  • Helix terminaiton
• Tyr HC2
  • Cross-links H and F helices

Effect of pH

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• Binding of O2 to Hb is dependent of pH
• Lower pH, affinity of Hb to O2 was reduced
• Higher pH, affinity of Hb to O2 was increased
• His 146 is responsible for this effect
  • Sensitive to pH and can be protonated and deprotonated reversibly
  • Forms a salt bridge with Asp 94 when protonated
  • When not protonated, the salt bridge with Asp 94 is broken
• Dramatic change in structure just from being protonated/deprotonated

Effect of CO2

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• CO2 binds to Hb-α-Val1-NH2
  • Froms carbamino hemoglobin
  • Carbanimo lysine on α subunit can form a salt bridge with Arge 141
    • Stablizes the T state
  • N-terminus of β subunit is also carbamylated
    • Allows effective transport of CO2

CO2 Transportation

• Most CO2 is transported as HCO3-
  • Hydration/Dehydration of CO2 doesn’t happen fast enough so it requires an enzyme: carbonic anhydrase
• Some is transported as HbCO2-
• Very little is transported as CO2
• pH at lung is higher than site of metabolism
  • At lung, favor binding of O2
  • At site of metabolism, favor disassociation of O2